Myelin is a fundamental component of mature neural networks that is affected in a large number of pathological conditions of the central nervous system (CNS). Critical for advancing knowledge about these conditions would be a better in vivo understanding of how oligodendrocytes and their respective myelin sheaths develop, are maintained throughout life and respond to injury. We have developed a new technique that allows high resolution label-less in vivo imaging of myelinated axons. This technique takes advantage of the high refractive index of lipid rich multilayered myelin and is based on multispectral confocal reflectance (MCORE) microscopy. Using MCORE we have obtained for the first time long- term images of the dynamics of cortical myelin on the cellular scale in a livig animal. Our preliminary data shows this technique as well as fluorescence imaging are a powerful set of tools that in combination provide a wealth of information about fine structural changes in oligodendrocytes and myelin in vivo. We demonstrate the feasibility to track the development of myelin pathology in a dysmyelinating mutant mouse and determine the temporal dynamics of demyelination after single oligodendrocyte ablation. We propose to use this powerful technique in combination with in vivo two-photon and confocal fluorescence imaging to address three fundamental questions concerning the plasticity and regeneration of neocortical myelin. First, we will determine the long-term in vivo dynamics of individual oligodendrocytes and myelinated axons from birth to death. Second, we will investigate how neuronal activity influences myelin formation and stability on individually active axons in vivo. Finally we will stuy the process of demyelination and remyelination after single oligodendrocyte ablation and determine how repeated ablation and neuronal activity influences the temporal dynamics of remyelination. Together these experiments will provide novel insight into the fundamental plasticity and regeneration capabilities of myelin and oligodendrocytes in relation to axonal activity throughout life.